MA14.02 RET Fusion Testing in Advanced Non-Small Cell Lung Carcinoma Patients: the RETING Study

Abstract

The approval of selective RET inhibitors for patients with advanced RET fusion positive non-small cell lung carcinoma (NSCLC) means that the importance of accurately identifying those patients has never been greater. Due to the co-existance of single-gene testing and next-generation sequencing (NGS), we hypothesized that a comparison of RET fusions testing assays is urgently needed. Along those lines, the performance of RET immunohistochemistry (IHC) and RET fluorescence in situ hybridization (FISH) has recently been challenged. Physicians across 17 hospitals contributed to identify patients with RET fusion positive NSCLC as part of routine clinical care. In addition, RET fusion negative samples from NSCLCs diagnosed at the referral institution were also included. All tumors underwent targeted RNA-based NGS (Oncomine Comprehensive Assay v3), break-apart RET FISH (Vysis) with an automated scanning system (BioView), and RET IHC (clone EPR2871, Abcam). The material available for all tumors had been formalin-fixed and paraffin-embedded. Only cases with enough tissue available (i.e. a minimum of 50 tumor cells, as per FISH test requirements) and sufficient sequencing coverage after NGS were included. FISH and IHC results were interpreted by using previously described criteria. The NGS result was used as the gold-standard. Analyses by the three assays was succesful in all 80 tumors. Thirty-seven RET-positive samples and 43 RET-negative NSCLCs were identified. Signet ring cells and psammomatous calcifications were frequently observed in RET-positive samples (in 30% and 27% of tumors, respectively). The most common partners were KIF5B (29/37, 78%), followed by CCDC6 (6/37, 16%). Other partners included NCOA4 (1/37, 3%) and AKAP13 (1/37, 3%). Thirty-five out of the 37 (95%) NGS-positive samples were FISH-positive (86% with a split pattern and the remaining 14% with a single 3’ signal pattern). Six of those FISH-positive cases (6/35, 17%) displayed very challenging split signals (≤2 signal diameter). The two FISH-negative samples were KIF5B-RET variants that showed a narrow split (≤1 signal diameter) in 36% and 38% of tumor cells, respectively. All 43 NGS-negative tumors were also FISH-negative. Regarding RET IHC, all NGS-positive cases showed cytoplasmic staining with a mean H-score of 210 (median 210, range 5-300). Within the NGS-negative cohort, cytoplasmic staining was observed in 8 cases (19%), with a mean H-score of 9 (median 0, range 0-150). Interestingly, the two RET FISH-negative but NGS-positive samples showed relevant IHC positivity (H-score of 165 and 255, respectively). The most frequent partner in this large multicenter series of RET fusion positive NSCLCs was KIF5B. The concordance between RNA-based NGS and FISH was high, with 0% failure rate. However, the interpretation of RET FISH is very challenging due to the frequent occurrence of narrow split signals (22% in this series). RET IHC should be investigated further as an alternative method to resolve an equivocal FISH or NGS result. Funding This study was mainly funded by Lilly. We also thank Instituto de Salud Carlos III (ISCIII) (Fondos FEDER and Plan Estatal I+D+I 2008–2011 [PI11/02866] and 2013–2016 [PI14-01176 y PI17-01001]) and the iLUNG Program (B2017/BMD-3884) from the Comunidad de Madrid.